European Journal of Medical Genetics 58 (2015) 71e74

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Clinical research

Eight novel mutations in MLC1 from 18 Iranian patients with megalencephalic leukoencephalopathy with subcortical cysts Ariana Kariminejad a, *, Ahmad Rajaee a, Mahmoud Reza Ashrafi b, Houman Alizadeh b, Seyed Hasan Tonekaboni c, Reza Azizi Malamiri d, Mohamad Ghofrani c, Parvaneh Karimzadeh c, Mohsen Molla Mohammadi e, Ali Baghalshooshtari f, Bita Bozorgmehr a, Mohamad Hasan Kariminejad a, N. Postma g, Truus E.M. Abbink g, Marjo S. van der Knaap g a

Kariminejad-Najmabadi Pathology & Genetics Center, Tehran, Iran Pediatrics Centre of Excellence, Department of Pediatric Neurology, Children’s Medical Centre, Tehran University of Medical Sciences, Tehran, Iran c Pediatric Neurology Research Center, SBMU, Tehran, Iran d Department of Paediatric Neurology, Golestan Medical, Educational, and Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran e Hazrat Fatemeh Masoumeh Hospital, Qom University of Medical Sciences, Qom, Iran f Ahvaz Behzisti Genetic Counseling Center, Ahvaz, Iran g Department of Child Neurology, VU University Medical Center, Amsterdam, The Netherlands b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 June 2014 Accepted 4 December 2014 Available online 11 December 2014

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) (MIM #604004) is a rare autosomal recessive neurological disorder characterized by macrocephaly, motor and cognitive decline, ataxia, spasticity and occasional seizures. Magnetic resonance imaging (MRI) shows diffusely abnormal and swollen white matter of the cerebral hemispheres and subcortical cysts in the anterior temporal and frontoparietal region. Mutations in MLC1(22q13.33) and GLIALCAM have been identified in patients with MLC. Mutations in MLC1 account for approximately 75% of the cases. MLC was suspected in eighteen Iranian patients from sixteen families based on positive clinical findings including macrocephaly beginning in the first year, neurocognitive deterioration, seizure or loss of consciousness after minor head trauma. All except two were born to consanguineous parents. Brain MRI images were compatible with MLC and confirmed the diagnosis. Sequencing of entire coding region of MLC1 was performed for seventeen patients and mutations in MLC1 were detected in all of them. Eight novel mutations and seven previously reported mutations were identified. This report shows that MLC is relatively common in Iranian population, as expected for rare diseases with high inbreeding, with a surprisingly high frequency of novel mutations. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: MLC1 Megalencephaly Leukoencephalopathy Mutations

1. Introduction Megalencephalic leukoencephalopathy with subcortical cysts (MLC; MIM604004) is an infantile-onset autosomal recessive neurological disorder [Goutieres et al., 1996; van der Knaap et al., 1995]. The disease was recognized independently in India and the Netherlands [Singhal et al., 1996; van der Knaap et al., 1995]. The disorder is characterized by macrocephaly present at birth or,

* Corresponding author. Kariminejad-Najmabadi Pathology & Genetics Center, #2, 4th St., Hasan Seyf St., Sanat Sq., Shahrak Gharb, Tehran, Iran. E-mail address: [email protected] (A. Kariminejad). http://dx.doi.org/10.1016/j.ejmg.2014.12.004 1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.

more often, during the first year of life, occasional seizures, and deterioration of motor function with ataxia and spasticity [Boor et al., 2006; Goutieres et al., 1996; Leegwater et al., 2001; Singhal et al., 1996; van der Knaap et al., 1995]. At birth, infants with MLC are healthy. Macrocephaly most commonly develops during the first year of life in the absence of other neurological signs [Goutieres et al., 1996; Singhal et al., 1996; Topcu et al., 1998; van der Knaap et al., 1995]. The disease is of variable severity in clinical symptoms and disease progression. Most children have a delay in walking without support and unstable gait [Goutières et al., 1996; Singhal et al., 1996; Topcu et al., 1998; van der Knaap et al., 1995]. The clinical signs slowly worsen with cerebellar ataxia and mild spasticity usually starting in early childhood or

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Table 1 Clinical and genetic findings of patients with MLC. Pt

Sex

Age

HC at birth cm

HC at examination

Seizure after head trauma

Onset of first seizure

Loss of consciousness after head trauma

Ataxia

Spasticity

Intention tremor

Dysmetria

Speech

1 2

F M

3.5 y 5y

35 (50th%) 35.5 (25e50th)

54.5 (>97%) 59.5 (>97%)

þ þ

2.5 y 5y

 þ

þ þ

e þ

þ þ

e þ

3 4

F M

11 m 3y

35 (50th%) 34 (25th%)

51 (>97%) 55.5 (>97%)

 

 

 

Not yet þ

Not yet 

Not yet þ

Not yet þ

5 6

F F

4y 6.5 y

36 (75th%) 36 (75th)

57 (>97%) 58 (>97%)

 þ

 3y

þ þ

þ þ

NE 

NE þ

NE þ

7

F

3yþ9m

31

56 (>97%)





þ









Nl Dysarthria Dyslalia Not yet Dysarthria Dyslalia Nl Dysarthria Dyslalia Nl

8 9 10 11 12 13 14

M F M F M F M

14 m 5y 3y 10.5 y 20 y 5.5 y 3y

33 (10th%) 35.5 (50e75th%) NE NL NE 34.5 (25e50th%) NE

51 (>97%) 55 (>97%) 57 (>97%) 57 (>97%) NE 57 (>97%) 58 (>97%)

þ   þ þ  

20 m  3y 4m  

 þ þ þ NE  þ

þ þ þ þ þ þ þ

   NE  

þ  þ þ NE  

þ  þ þ NE þ 

Dysarthria Nl Dysarthria Nl NE Dyslalia Nl

15 16 17

F F M

5.5 y 6y 6y

33 (8 m) 36 (75th%) Nl

55.5 (>97%) 56.5 (>97%) 61 (>97%)

  þ

  3y

þ þ 

þ  þ

 e þ

þ e þ

þ  þ

18

M

6m

35 cm (50th%)

50 cm (>97th%)









e

e

NE

Nl Dysarthria Dysarthria, Dyslalia NE

Abbreviations F, female; HC, head circumference; m, month; M, male; NE, no evaluated; Nl, normal; SCC, subcortical cyst; SWM, swollen white matter.

later [Goutières et al., 1996; Singhal et al., 1996; Topcu et al., 1998; van der Knaap et al., 1995]. By the age of 13e19, patients are usually wheelchair-bound [van der Knaap et al., 2012]. Cognitive skills are normal or mildly decreased [Goutières et al., 1996; Singhal et al., 1996; Topcu et al., 1998; van der Knaap et al., 1995]. Most individuals have occasional epileptic seizures that are usually easily controlled with medication [van der Knaap et al., 1995], but status epilepticus can occur [Mejaski-Bosnjak et al., 1997; Singhal et al., 1996]. Seizures or status epilepticus, prolonged unconsciousness or acute motor deterioration with gradual improvement can occur after minor head trauma [BenZeev et al., 2001; Bugiani et al., 2003; Riel-Romero et al., 2005]. No systematic study of the lifespan of patients with MLC has been performed. Some patients die in their teens or twenties, whereas others stay alive into their forties [Pascual-Castroviejo et al., 2005; Saijo et al., 2003; Singhal et al., 1996; Singhal et al., 2003]. There are differences in disease severity among patients with the same mutation [Gorospe et al., 2004; Goutières et al., 1996; Singhal 2005; Singhal et al., 2003] and sibling patients [PascualCastroviejo et al., 2005; Patrono et al., 2003; Singhal 2005; Singhal et al., 2003]. Magnetic resonance imaging (MRI) shows diffusely abnormal and swollen white matter of the cerebral hemispheres and subcortical cysts in the anterior temporal and often also frontoparietal region. Large temporal cysts are the radiological hallmark of this disorder [van der Knaap et al., 1995]. This disease is histopathologically characterized by myelin splitting and intramyelinic vacuole formation [Boor et al., 2006]. MLC is caused by mutations in MLC1 or GLIALCAM. MLC1 is an oligomeric membrane protein that is expressed almost exclusively in the brain. The first gene mutated in MLC (MLC1) was identified in 2001 [Leegwater et al., 2001]. It contains 12 exons, with a start codon in exon 2 and a 30 untranslated region (UTR) of 2.2 kb MLC1 is located on chromosome 22qtel [Topcu

et al., 2000]. Mutations in this gene have been identified in approximately 75% of the MLC patients [Boor et al., 2006]. There is only one report of an Iranian patient with genetic confirmative diagnosis of MLC [Ashrafi et al., 2009]. In this study, we sequenced the entire coding region of MLC1 in seventeen Iranian patients, including the previously reported patient and were able to identify mutations in all seventeen. Eight mutations were novel and seven were previously reported. This report shows that MLC is relatively common in Iranian population, as expected for rare diseases with high inbreeding, with a surprisingly high frequency of novel mutations. 2. Material and methods We selected our patients on the basis of clinical criteria including early onset macrocephaly, normal or almost normal initial motor and cognitive milestones, slow deterioration of motor followed by cognitive function and normal metabolic studies. MRI was requested for all these patients. In two families where there were two affected siblings, MRI of one sibling was requested. Diagnosis was confirmed by MRI findings. Blood samples were collected from seventeen of these patients and their parents. DNA was extracted according to a standard procedure [Miller et al., 1988]. Sequencing of the MLC1 gene was performed, as reported before [Leegwater et al., 2001]. 3. Results Clinical findings and mutations are summarized in Table 1. MRI findings were consistent in all sixteen patients showing diffusely abnormal and swollen white matter of the cerebral hemispheres, and the presence of subcortical cysts in the anterior temporal and frontoparietal region (Fig. 1A, B).

A. Kariminejad et al. / European Journal of Medical Genetics 58 (2015) 71e74

Develop-mental Onset of Mutation Onset of Onset of delay macrocephaly motor cognitive deterioration deterioration

Mutation type

73

Mutation status Parents Brain MRI consanguineous Findings

Not yet

not yet

Mild

7m

c.353c > T,p.Thr 118Met homozygous

Missense

Reported

þ

SWM, SCC

Not yet

4y



3m

c.136delT,p.Cys46AlafsX11 homozygous

Frameshift

Reported

þ

SWM,SCC

Not yet 2yþ8m

Not yet 2yþ8m

þ þ

7m 7m

c.322-6T > C homozygous c.250C > A, p.Arg84Ser homozygous

Splicing Missense

Novel Novel

þ þ

SWM, SCC SWM, SCC

NE Not yet

10 m 3y

 þ

4m 7m

c.177 þ 1G > T splice defect at protein level Splicing C.459delG, p.Glu153AspfsX6 Frameshift

Reported Novel

þ þ

SWM,SCC SWM, SCC

Not yet

Not yet

þ

4e5 m

Not Not Not Not NE Not Not

yet yet

Not yet Not yet Not yet 4y NE 4y 2.5 y

   þ NE  Speech delay

8 8 6 3 4 4 2

c.135delC, p.Cys46Alafs11 & c.449-455del,p.Leu151_Leu152del, Compound-heterozygous c.268T > G, p.Cys90Gly homozygous c.736delA, p.Ser246Valfs*16homozygous c.736delA,p.Ser246Valfs*16homozygous c.278C > T, p.Ser93Leu homozygous c.976T > C/p.Cys326Arg homozygous c.976T > C/p.Cys326Arg homozygous c.42C > T homozygous (NOVEL*) (Functional study)

Not yet Not yet Not yet

3 Not yet 30 m

 No Yes

3m 6m 5m

Not yet

Not yet



4m

yet yet yet yet

m m m m m m y

Frameshift/deletion Reported/Novel 

SWM,SCC

Missense Frameshift Frameshift Missense Missense Missense NE

Novel Novel Novel Reported Reported Reported Novel

þ þ þ þ þ þ þ

SWM, SCC SWM, SCC SWM, SCC

c.136delT,p. Cys46AlafsX12 c.423þ1G > A, p.homozygous

Frameshift Splicing

Reported Reported

 þ þ

SWM, SCC SWM, SCC SWM, SCC

c.701G > A, p. Trp234X homozygous

Premature stop in Protein level

Novel

þ

SWM, SCC

We suspected a diagnosis of MLC in eighteen patients on the basis of typical clinical picture and MRI findings. Seventeen gave consent for genetic testing and we were able to identify eight novel mutations in nine patients, of which two siblings had the same novel mutation, seven of which were homozygous, and one was compound-heterozygous. The remaining eight patients had seven different, previously reported mutations, of which two siblings and two unrelated patients had the same mutation. The previously reported mutations were as follows: c.136delT in exon 2 leading to a frameshift and a premature stop at protein level, two splice-site mutations, c.177 þ 1G > T and c.423 þ 1G > A, three missense mutations c.353C > T, c.278C > T, c.976T > C and one deletion

SWM, SCC

SWM, SCC

c.449-455del. Three novel missense mutations, c.250C > A, c.268T > G, c.701G > A, and one novel splice site mutation, c.3226T > C, were identified. Three novel frameshift mutations were identified, c.459delG, causing a frameshift in the open reading frame, leading to a premature stop, and c.135del.C leading to a frameshift and truncation of the protein and c.736delA leading to a premature stop at protein level. These mutations were not found in >400 control chromosomes, nor in the 1000 genome database (http://browser.1000genomes.org/index.html). We found a c.42C > T mutation in one of our patients. This mutation creates a new start codon in the 50 untranslated region of the MLC1 mRNA. The Kozak sequence surrounding the new start codon is adequate:

Fig. 1. (A) Axial brain MRI of case 14 presenting with mild neurologic developmental delay, revealed diffuse abnormal white matter signal-typically high in T2-SE sequence suggesting diffuse white matter swelling. (B) Coronal brain MRI, T2-SE sequence of the same patient showed bilateral subcortical anterior temporal cysts formation isosignal with cerebrospinal fluid in the context of diffuse white matter abnormal signal, high in T2-WI. The cysts are present in the subcortical superior temporal gyri location beneath the opercular region bilaterally. Cystic nature of these lesions was confirmed by FLAIR sequence (not shown in these pictures).

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there is a guanine at the 3 position which is the most important residue of the Kozak motif. The upstream open reading frame created by the new start codon is out of frame and overlaps with the MLC1 open reading frame. These features together indicate that the variant reduces the synthesis of MLC1 protein, as has been extensively described for other mRNAs. If this new start codon would be used, the resulting protein would be out of frame with the main reading frame and would lead to a premature stop at protein level. The same mutation has not been found in >400 control chromosomes nor in the 1000 genome database, making it unlikely that it represents a common polymorphism. This mutation has not been published, but has been observed in a homozygous state in another MLC patient [van der Knaap, personal communication]. 4. Discussion In this study eighteen patients from 16 families suspected with MLC based on brain MRI findings were evaluated. Clinical features in our patients were similar to reports from this condition. The patients’ age ranged from six months to twenty years with the median age of three years. We had clinical information from 17/18 patients. All of the seventeen had onset of macrocephaly before the age of one year and 8/17 patients had developmental delay. Onset of motor deterioration varied from one year to four years in nine patients but had not initiated in eight patients ranging in age from six months to six years. Cognitive deterioration was seen in only three patients, in one time of onset was not known and was at 2 years and 8 months, and 4.5 years in the other two. Seventeen patients agreed for genetic testing and we were able to identify mutations in all 17 patients. We identified 8 novel and 7 previously reported mutations, which means a 53% rate of novel mutations. The only recurring mutation identified in our patients was c.136delT in exon 2 causing a frameshift and premature stop at the protein level; otherwise all other mutations were found only in one family. This is different from the Indian population where about 73%of the patients have the same c.135_136insC, or even in the Chinese population where 23.7% have a mutation in c.772-1G > C, indicating that in spite of the relatively high frequency of this rare disease in Iran, it is not caused by a founder effect [Shukla et al., 2011; Wang et al., 2011]. There have been reports of MLC1 in different ethnicities, including Indian, Turkish and Chinese population from Asia. The Chinese reported the highest rate of novel mutations (70%) [Wang et al., 2011]. This was expected as there was not any previous report from the Chinese population or neighboring countries. The study from the Turkish population and Indian reported a 5/13 (38%) and 2/37 (5%) rate of novel mutations respectively [Shukla et al., 2011; Yuzbasioglu et al., 2011]. It is interesting that in spite of several reports from the neighboring countries of Iran, the Iranian population shows a high rate of novel mutations suggesting that the mutations in Iran are unique to its population. All seventeen patients that underwent genetic testing showed mutations in MLC1 gene, and that no GLIALCAM mutations were identified. In previous studies MLC1 mutations were found in only 75% of patients with clinical and MRI features of MLC. As the phenotype and MRI findings in patients with MLC1 and GLIALCAM mutations are similar, we cannot say that there was a bias in patient selection. Considering the absence of GLIALCAM mutated patients in our study we could

hypothesize that in the Iranian population a higher percentage of MLC patients have MLC1 mutations than other communities. Of course, a larger group of Iranian patients need to be evaluated to reach this conclusion.

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